1. Field of the Invention
The present invention relates to a method for classifying and counting a specific cell in the field of clinical testing or cell study. More particularly, it relates to a method for labeling leukocytes contained in a blood sample.
2. Related Art
Generally, in a case of determining a specific cell contained in a biological sample such as blood and urine, containing a variety cells, if the appearance of the cells (such as size and shape) is significantly different among them, it is not difficult to distinguish one from others by using a conventional microscope of transmitted light type. For example, since erythrocytes, leukocytes and blood platelets contained in blood are different in size and shape from each other, they can be easily distinguished.
In contrast, it is difficult to classify cells having a similar appearance with each other. For example, lymphocytes, monocytes, neutrophils, eosinophils and basophils; which belong to subclasses of leukocytes; are not easily classified. In general laboratories, components contained in cells such as a nucleus, granules, cytoplasm and endoenzyme in a leukocyte are stained with an appropriate dyeing solution to visualize the existence of the components contained in the cell and their amount and localization, thereby classifying the cells and providing for their counting.
In other case, a device for measuring cell volume or a device for measuring a scattered light or fluorescence polarized light from a cell is utilized instead of using a microscope. Cells are detected by such a device by measuring the distinctive signals depending on the characteristics of the respective cell in order to classify the cells. If the distinctive signals are not obtained only by using such device, the cells are subjected to a suitable treatment so as to obtain available signals which are different among the cells. Examples of such treatments include a method for lysing cells other than a specific objective cell with a suitable cell lysing agent or a method for detecting the difference in volume of cells, or optical difference of scattered light, fluorescent light, absorbency, or the like. In order to detect the optical difference by using a scattered light, fluorescent light, absorbency and the like, it is preferred to combine a suitable labeling substance such as a dye with at least one component in the cell.
It is known that components contained in cells are separated from the outside by a membrane, called a cell membrane, such that the components do not leak from the cell. However, the cell membrane does not work for complete sealing from the outside. Rather, some substances necessary for survival of the cell are incorporated into the cell and waste products unnecessary for the cell are excreted from the cell. Thus, the cell membrane selects substances accurately and ingeniously to allow substances to pass through. Substances inside and outside the cell pass through various channels for ion passage provided in the membrane and pass through a lipid bilayer by dispersion. Such selective movement of substances continues so long as the cell is alive, but when the cell dies, the selective operation is lost. In accordance with this phenomena, a method for distinguishing live cells from dead cells is known as a dye-exclusion test, in which a dye which can invade into a damaged cell but can not invade into an undamaged cell is used as a labeling substance. An example of the well-known dye-exclusion test is to use a dye such as trypan blue and eosin.
However, such a known method is not always carried out easily. That is, it is not possible to incorporate a labeling substance such as a dye into cells because cells naturally refuse the invasion of unnecessary substances to themselves, or even if it is possible it takes a long time. Generally, when cells are stained, various fixations are performed on the cells to inhibit the invasion function or prevent deformation of the cell and leak of components contained in the cell. In general, aldehydes such as formalin and glutaraldehyde, or methanol and acetone are used as a fixing agent. However, the use of such these fixing agents suffers from drawbacks such as involving a risk in handling and necessity for detoxifying waste water because of their toxicity, which is not a low, or requiring a long time, and a troublesome process for fixation.
On the other hand, a method for generating optical difference without fixing is also known, in which the selective substance exclusion function of the cell membrane is inhibited to assist in combing the cell components with a dye which generally can not pass through the cell membrane. A well known example comprises the steps of lysing the cell membrane and cytoplasm with a nonionic surfactant such as Triton X-100; staining the remaining nuclei with propidium iodide, ethidium bromide, etc. to prepare a test sample; followed by measuring a fluorescence light of the cell by a flow cytometer, microspectrophotometer, etc. and determining an amount of DNA. However, Triton X-100 lyses cell membranes and cytoplasms, and it also damage nuclei to no small extent, so that the data of DNA amount is made inaccurate. Accordingly, stabilization of nuclei, for example adding a stabilizer such as spermidine [N-(3-aminopropyl-1,4-butanediamine] or conducting some other fixation is necessitated in this method. Moreover, as the nuclei are naked, it is not possible to precisely classify leukocytes into each subclass by using scattered light and the like.
It is known to use two kinds of quaternary ammonium salt type surfactants for classifying leukocytes into two types i.e., mononucleocyte group and granulocyte group and analyzing them (for example disclosed in WO84/03771 and W084/02777). However, the concentration of the surfactants is high, for example, 40 to 70 g/l for one quaternary ammonium salt type surfactant and 2 to 7 g/l for another, which destroys leukocytes themselves and results in naked nuclei, so that it was not possible to classify and counting leukocytes by measuring optical difference.
Japanese Laid-Open Patent Application 88896/1986 discloses a method and a reagent for classifying and counting basophils which belong to leukocytes by using a water soluble surfactant in the limited pH range of 1.8 to 2.3 with a blood sample to generate an optical difference of the basophils. However, the surfactant is also used at a high concentration of 10 to 20 g/l and the method fails to disclose the use of a labeling substance.